The detection and quantitative determination of hydrogen peroxide and compounds yielding hydrogen peroxide as a result of chemical or enzymatic reactions are of importance in many areas. For example, they are important in the detection of hydrogen peroxide produced in the enzymatic assay of chemical or biological substances (sometimes called analytes) such as glucose, cholesterol, uric acid, lipase, triglycerides, creatine kinase, etc. in the presence of oxygen. The quantity of analyte present in a test sample is determinable from the amount of hydrogen peroxide produced and detected.
Known compositions for detecting or quantifying hydrogen peroxide in such assays generally comprise a substance having peroxidative activity, e.g. peroxidase, and a material which undergoes a detectable change (e.g. oxidation to a color dye) in the presence of hydrogen peroxide and the peroxidative substance. Various materials which undergo such a detectable change include monoamines, diamines, phenols, leuco dyes and other known dyes or dye formers.
Hydrogen peroxide detection has also been accomplished by the reaction of a color-forming coupler and an oxidizable color developing compound, e.g. 4-aminoantipyrine in the presence of peroxidase. Color-forming couplers which have been used for this purpose include N-substituted anilines, such as those described in U.S. Pat. No. 4,251,629 (issued Feb. 17, 1981 to Yamanisi et al), U.S. Pat. No. 4,260,679 (issued Apr. 7, 1981 to Tsuda et al) and U.S. Pat. No. 4,396,714 (issued Aug. 2, 1983 to Maeda et al). Some of the anilines described in these references have solubilizing groups, e.g. hydroxy or sulfo groups, attached to the nitrogen atom.
Although the dye-providing materials of the prior art are, in general, useful as indicators for hydrogen peroxide determination, there are instances when the concentration of hydrogen peroxide to be analyzed is too low to produce sufficient detectable color from such indicators. In some instances, this shortcoming can be overcome by using increased amounts of dye-providing materials. However, where the analyte concentration is initially low, or where high dilution of the test sample is required, such materials may still provide insufficient detectable color in such instances.
Such a problem of low analyte concentration is particularly acute when analyte determination is attempted with a dry analytical element, e.g. with the commercially successful elements described in U.S. Pat. No. 3,992,158 (issued Nov. 16, 1976 to Przybylowicz et al). In such instances, the indicator or reagent layer present in such elements is very thin, and the dye-providing material concentration is necessarily low. Hence, the density of the color formed from low level analytes, or even from abnormally low concentrations of high level analytes, can be rather low. However, it would be desirable to use such elements to assay for analytes present at low concentrations.
Hence, there is a continuing need in the art for a means to detect low concentrations of either hydrogen peroxide or analytes which react to produce hydrogen peroxide.